Streptomyces species synthesize more than 70 medically important antibiotics as well as antitumor and antiparasitic agents. These compounds are produced as secondary metabolites during nutritional limitation and the initiation of sporulation. However, the mechanisms which regulate the initiation of secondary metabolism and sporulation in Streptomyces are not understood. The synthesis of both glutamine synthetase (GS), a key enzyme in nitrogen assimilation, and the antibiotic, actinorhodin, are increased during nitrogen limitation in S. coelicolor. Investigations of the regulation of GS in other bacterial systems have identified global regulatory networks which activate gene expression in response to nitrogen availability. The GS gene will be used as a handle for studying regulatory mechanisms responsible for alterations in gene expression during nitrogen limitation in Streptomyces. Two approaches will be used to investigate the regulation of the GS gene in S. coelicolor. First, the S. coelicolor GS structural gene (glnA) which has been cloned in this laboratory will be physically characterized to determine the glnA promoter and coding region. Secondly, factors involved in GS regulation will be identified genetically by the isolation of mutants altered in the ability to synthesize GS. The glnA cloned DNA will then be used to study the interactions between the glnA promoter and the genetically identified GS regulatory proteins, RNA polymerase and small nitrogen-containing compounds. An understanding of the mechanisms which mediate nitrogen regulation of GS in Streptomyces will allow rational strategies to be used for the increased production of antibiotics and other secondary metabolites, and the isolation of novel secondary metabolites. Furthermore, it should be possible to develop a better understanding of the regulatory mechanisms involved in the initiation of secondary metabolism and sporulation in Streptomyces.